NAPCLI: Non-conventional approaches for peptidoglycan cross-linking inhibition

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Peptidoglycan (PG) is an attractive and validated target for antibacterial drug development for two
main reasons. First, it is an essential and unique bacterial cell wall polymer with no counterpart in
human cells, minimizing the risk of drug toxicity. Second, the essential PG synthases are exposed at
the outer surface of the cytoplasmic membrane, making them highly accessible for antibiotic
inhibition. Formation of the PG network requires glycosyltransferases for glycan chain elongation
and transpeptidases for peptide cross-linking. Transpeptidation involves two stem peptides that act
as acyl donor and acceptor substrates, respectively. The acyl donor site is targeted by the
ß-lactams, which form covalent adducts, and this interaction is well characterized. In contrast,
nothing is known on the interaction of the transpeptidases with the acceptor substrate. To combat
the erosion of the activity of ß-lactams, we propose to identify additional drugable sites in the
transpeptidases, including the acceptor binding site, and develop lead antibacterial agents acting
on these sites. Our first objective is to characterize the mode of recognition of the acyl acceptor by
transpeptidases and identify compounds blocking the binding of this substrate. We will use NMR
spectroscopy to map the acceptor site and develop specific inhibitors based on modeling and
virtual screening. Our second objective is to identify the partners of transpeptidases that regulate
the coordinated elongation of glycan chains and cross-linking of stem peptides. This will allow us to
select additional drugable sites in transpeptidases and associated proteins within the PG
polymerization complexes. We will map key interactions by FRET analyses in live bacteria producing
fluorescent proteins and by in vitro transpeptidase/glycosyltransferase assays in complexes
obtained by tandem-affinity purification. Microfluidic cultures and time-lapse microscopy will assess
the impact of inhibitors on cell division and viability. The interaction of lead compounds with their
targets will be characterized by X-ray crystallography. These complementary approaches will
enable the consortium to develop novel strategies for transpeptidase inhibition and obtain leads
active against ß-lactam-resistant bacteria.